Cancer cell metastasis requires cellular capacity to 1) detach from a primary tumor, 2) migrate and invade through local tissues, 3) translocate to distant sites in the body (via lymph or blood), 4) colonize a foreign site, and 5) grow and survive in this foreign environment. All of these behaviors are linked to cell adhesions. Cell adhesions control the physical interactions of cells with their microenvironment. Cell adhesions also initiate signals that dictate tumor cell growth, death, and differentiation.
Various cancer cells, including breast cancer cells, are known to exhibit altered cell adhesion. As compared to normal breast epithelia, transformed human breast epithelial cells have decreased cell—cell contacts and increased interactions with the surrounding extracellular matrix. These changes facilitate increased detachment and migration of cancer cells away from cell colonies and are directly linked with alteration in tyrosine phosphorylation of cell membrane proteins. Tyrosine phosphorylation is a potent form of cell signal transduction, and alteration in levels of tyrosine phosphorylation is believed to be important for tumor cell invasiveness. Thus, regulation of tyrosine phosphorylation represents a promising target for therapeutic intervention against metastatic cancer. Tyrosine phosphorylation is controlled by cell membrane tyrosine kinases, and increased expression of tyrosine kinases is known to occur in metastatic cancer cells.
Identification of increase expression of cell membrane tyrosine kinases would aid in the diagnosis and treatment of metastatic diseases. One such tyrosine kinase in EphA2. A member of the Eph family of tyrosine kinases known as Ephrins, EphA2 is a transmembrane receptor tyrosine kinase with a cell-bound ligand. Although cloned a decade ago, see Lindberg, R. A. and Hunter, T., “cDNA Cloning and Characterization of Eck, an Epithelial Cell Receptor Protein-tyrosine Kinase in the Eph/elk Family of Protein Kinases,” Mol, Cell. Biol. 10(12), 6316–6324 (1990), rather little is known about EphA2 function.
To facilitate research on EphA2, an improved method for generating a panel of monoclonal antibodies specific for tyrosine phosphorylated proteins has been developed. Using this method, a multiplicity of EphA2 recognizing monoclonal antibodies has been generated. These antibodies have been used to show that EphA2 is overexpressed in metastatic breast, lung, colon, and prostate cells. Because EphA2 is expressed differently in normal and metastatic cells, EphA2-specific antibodies are useful in the diagnosis of metastatic disease. Antibodies produced by one particular hybridoma recognize an intracellular epitope of EphA2 and have been shown to be highly specific in binding to EphA2.
Thus, one aspect of this invention is a compound which specifically binds to an intracellular epitope of EphA2. In a preferred embodiment, the compound is an antibody specific for a domain of the EphA2 protein. However, natural or artificial ligands, peptides, anti-sense, ATP analogues, or other small molecules capable of specifically targeting EphA2 may be employed. A second aspect of this invention is a method for generating antibodies which recognize EphA2 intracellular epitopes. Another aspect of this invention is the use of EphA2-specific antibodies in the diagnosis of metastatic disease. An additional aspect of this invention is a diagnostic reagent specific for detecting EphA2, any fragment thereof, or DNA or RNA coding for the EphA2 protein.
Additional features of the present invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.